The technology for producing cell sheets is important in the field of tissue engineering; however, it has some drawbacks.
First, it is difficult to detach a cell sheet cultured on a cell culture substrate from the substrate, which is problematic. In addition, even if such cell sheet can be detached from the substrate, the cell sheet contracts after detachment. In such case, it is difficult to handle the obtained cell sheet in the subsequent steps, which is also problematic.
For example, Patent Document 1 discloses a technique comprising providing a temperature responsive polymer to the surface of a cell culture base substrate in order to facilitate the detachment of a cell sheet, culturing a cell sheet thereon, and changing the temperature so as to allow the detachment of the cell sheet. However, the technology is not applicable to some cell species that are difficult to detach. Further, the problem of contraction of a detached cell sheet has not been solved by the above technique. Furthermore, it is difficult to produce a thick cell sheet by the above technique, which is also problematic.
Patent Document 2 discloses a technique for coating a substrate surface with fibrin glue in order to facilitate the detachment of a cell sheet. Patent Document 2 describes that fibrin glue is degraded by an enzyme contained in cells and disappears such that cells binding to each other and forming a cell sheet become suspended, facilitating the detachment of the cell sheet. However, in Patent Document 2, a scraper is used to detach the cell sheet. Therefore, it cannot be said that a sufficient degree of detachability has been achieved in the above case. Further, it is thought that some cell species cannot cause sufficient degradation of fibrin glue. In addition, the problem of contraction of a detached cell sheet has not been solved yet.
In addition to be above, techniques for facilitating the detachment of a cell sheet by controlling adhesiveness between a cell sheet and a culture substrate have been disclosed (see Patent Documents 3 to 6, etc). However, each one of the above techniques is not applicable for some cell species. In addition, the problem of cell sheet contraction cannot be solved by the above techniques.
Further, there are advanced techniques whereby a multilayer cell sheet can be formed. In the field of tissue engineering, techniques for regenerating thin tissues such as skin tissue and tissues with low cell content such as cartilage tissue have already been used in practice. In addition, many therapeutic methods comprising injecting therapeutically effective cells into lesions are being examined in clinical trials. However, tissue engineering technology has not been established for tissues with higher cell contents (with many examples of such tissues existing). In particular, in order to artificially produce tissues with high oxygen demand (tissues containing blood vessel networks) and tissues comprising parenchymal cells such as heart tissue and liver tissue, it is necessary to adjust the cell sheet thickness to at least 200 μm. However, if usual cell sheets are laminated to the above or a greater thickness, it becomes impossible to supply oxygen in a sufficient amount to cells contained inside the obtained laminate, resulting in necrosis. This is problematic.
Hitherto, in order to supply oxygen to cells, many techniques for forming a cell tissue by seeding cells on a porous scaffold (carrier) comprising, for example, a bioabsorbable material have been examined. However, in the cases of such techniques, it is difficult to achieve uniform distribution of cells inside a scaffold. In addition, a transplanted tissue tends to be fibrosed, which is problematic.
Further, when a multilayer cell sheet is produced, such cell sheet is more likely to contract than single-layer cell sheets, which is also problematic.
Meanwhile, Patent Documents 7 and 8 disclose techniques for culturing cells on a vitrified hydrogel thin film having improved shape retention. Hydrogel thin films comprising collagen and the like described in Patent Documents 7 and 8 are physically strong. Therefore, it is thought that cell sheets cultured on such thin films are unlikely to contract. However, in the above cases, a very thick hydrogel layer is used. Accordingly, even if it is possible to laminate a plurality of cell layers comprising hydrogel layers obtained in Patent Document 7 or 8, an inter-cell-layer network is unlikely to be formed because of large gaps between overlapping cell layers. In addition, it is difficult to obtain inter-cell-layer paracrine interaction that is induced by diffusion of liquid factors or the like. Consequently, the above techniques are not appropriate for production of regenerative tissue.
Patent Document 1: WO2002/008387
Patent Document 2: WO2005/028638
Patent Document 3: JP Patent Publication (Kokai) No. 2006-346292 A
Patent Document 4: JP Patent Publication (Kokai) No. 2006-94799 A
Patent Document 5: JP Patent Publication (Kokai) No. 2005-261292 A
Patent Document 6: JP Patent Publication (Kokai) No. 2006-296896 A
Patent Document 7: WO2005/014774
Patent Document 8: JP Patent Publication (Kokai) No. 8-228768 A (1996)